Method for diagnosing hepatic fibrosis

ABSTRACT

The present invention concerns methods, in particular in vitro methods, for diagnosing liver fibrosis in a subject suffering from obesity, or for selecting a subject suffering from obesity for liver biopsy or for treatment. The present invention also pertains to methods for screening a probiotic, a prebiotic, a chemical compound or a biological compound suitable for preventing and/or treating liver fibrosis.

The present invention concerns methods for diagnosing liver fibrosis ina subject suffering from obesity, or for selecting a subject sufferingfrom obesity for liver biopsy or for treatment. The present inventionalso pertains to methods for screening a probiotic, a prebiotic, achemical compound or a biological compound suitable for preventingand/or treating liver fibrosis.

The liver is the first extra-intestinal organ that encounters venousblood from the small and large intestines via the portal vein. In ahealthy organism only minor quantities of translocated bacterialproducts reach the liver. In general, the hepatic immune systemtolerates these bacterial products, avoiding harmful responses, aphenomenon known as ‘liver tolerance’. In this respect, the liver notonly consists of parenchymal hepatocytes, but also containsnon-parenchymal cells including immune and non-immune cells. Members ofthe hepatic immune system are resident liver tissue macrophages (Kupffercells), natural killer (NK) cells, NKT cells, T cells and B cells. Thesecell types strictly regulate the liver immune system including livertolerance.

Several lines of evidence suggest that bacterial translocation plays asignificant role in alcoholic liver diseases. It has been observed thatimpairment of the function of intestinal tight junctions and bacterialproliferation in the gut induced by alcohol and/or its metabolites, suchas acetaldehyde, enhance bacterial translocation to the liver. This inturn leads to activation of immune cells, including Kupffer cells, andthe release of various pro-inflammatory cytokines and chemokines. Inaddition, it has been observed that there is reduced intestinalexpression of the antimicrobial proteins Reg3b and Reg3g in animals andpatients with chronic ethanol consumption, which suggests thatethanol-induced gut dysbiosis is mediated by the deregulation of theexpression of antimicrobial molecules.

In non-alcoholic steatohepatitis (NASH), evidence of the role ofbacterial translocation into the liver also exists. Firstly, the role ofgut flora in obesity has been demonstrated. When wild-type germ-freemice fed a standard chow diet were colonized with a microbiota harvestedfrom ob/ob or lean donors, adiposity in recipients of the “obese”microbiota increased more than in recipients of a “lean” microbiota,indicating a crucial role of the microbiota in obesity and hence fattyliver disease. However, to the inventors' knowledge, no correlation hasever been established between bacterial translocation into the liver, oreven into the blood circulation, of a subject and the presence of liverfibrosis in said subject.

Liver fibrosis is a significant health problem with a worldwidemortality attributable to cirrhosis and primary liver cancer of around1.5 million deaths per year. Cirrhosis is the last stage of fibrosiswhich occurs mainly in response to viral and toxic-metabolic insults.The most common causes of fibrosis progression are chronic hepatitis C,chronic hepatitis B, alcoholic liver disease and non-alcoholic fattyliver disease. Liver fibrosis is treatable, even at the cirrhotic stage,mainly using anti-viral treatments for hepatitis C and B, but also byreducing alcohol consumption and improving overweight, diabetes andmetabolic factors for non-alcoholic fatty liver disease.

Therefore, screening for liver fibrosis is desirable. Liver biopsy isthe standard for diagnosing hepatic fibrosis and for diagnosing theunderlying liver disorder causing fibrosis. However, liver biopsy isinvasive, resulting in a 10 to 20% risk of minor complications (e.g.postprocedural pain) and a 0.5 to 1% risk of serious complications (e.g.significant bleeding). Also, liver biopsy is limited by sampling errorand imperfect inter-observer agreement in interpretation of histologicfindings. Thus, liver biopsy may not always be done, and alternativenon-invasive methods for diagnosing liver fibrosis are highly needed.

The present invention first arises from the unexpected finding by theinventors that the blood concentration of bacterial 16S rDNA isspecifically correlated to the presence of liver fibrosis in patientssuffering from obesity, whereas it is not correlated to other kind offatty liver diseases, such as e.g. liver steatosis. The bloodconcentration of bacterial 16S rDNA may thus be useful in the diagnosisof liver fibrosis in patients suffering from obesity.

The present invention thus relates to a method, in particular an invitro method, for diagnosing liver fibrosis in a subject suffering fromobesity, said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), diagnosing liverfibrosis in the subject suffering from obesity.

The present invention also concerns a method, in particular an in vitromethod, for selecting a subject suffering from obesity for liver biopsy,said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo liver biopsy.

According to another aspect, the invention relates to an in vitro methodfor selecting a subject suffering from obesity for treatment regimentargeting liver fibrosis and/or its complications, said methodcomprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo treatment regimen targetingliver fibrosis and/or its complications.

Another aspect of the present invention is a method for treating anobese subject suffering from liver fibrosis, said method comprising thesteps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject;

b) based on the result of the measurement in step a1), diagnosing liverfibrosis in the subject suffering from obesity; and

c) administering to the subject a treatment targeting liver fibrosis, ifliver fibrosis has been diagnosed in step b).

The invention further pertains to a method for treating a subjectsuffering from obesity and from liver fibrosis and/or its complications,said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject;

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo drug treatment targeting liverfibrosis and/or its complications; and

c) administering to the subject selected in step b) a drug treatmenttargeting liver fibrosis and/or its complications.

The invention also relates to in vitro method for monitoring theresponsiveness of a patient suffering from obesity and from liverfibrosis and/or its complications to a drug treatment targeting liverfibrosis and/or its complications, said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), monitoring theresponsiveness of the patient to said drug treatment.

The blood concentration of bacterial 16S rDNA reflects the cause of thedisease rather than its consequence, and may therefore be useful as acompanion biomarker to therapeutic strategies targeting gut microbiota,such as pre and probiotics.

Accordingly, the present invention finally pertains to a method forscreening a probiotic, a prebiotic, a chemical compound or a biologicalcompound suitable for preventing and/or treating liver fibrosis,comprising the steps of:

measuring the concentration of bacterial 16S rDNA, or the ratio of thequantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of an obese subject suffering from liver fibrosis whohas been treated with the candidate probiotic, prebiotic, chemicalcompound or biological compound; and

comparing said concentration, or said ratio, with that of a controlobese subject suffering from liver fibrosis who has not been treatedwith said candidate probiotic, prebiotic, chemical compound orbiological compound;

wherein a higher concentration, or a higher ratio, measured in thebiological sample of the control subject than in the biological sampleof the subject treated with the candidate probiotic, prebiotic, chemicalcompound or biological compound, indicates that said candidateprobiotic, prebiotic, chemical compound or biological compound issuitable for preventing and/or treating liver fibrosis.

DETAILED DESCRIPTION OF THE INVENTION Fibrosis

As used herein, the terms “fibrosis”, “liver fibrosis” or “hepaticfibrosis” refer to a medical condition in which excessive connectivetissue accumulates in the liver; this tissue represents scarring inresponse to chronic, repeated liver cell injury. Commonly, fibrosisprogresses, disrupting hepatic architecture and eventually function, asregenerating hepatocytes attempt to replace and repair damaged tissue.

Subject

In the context of the present invention, a “subject” denotes a human ornon-human mammal, such as a rodent (rat, mouse, rabbit), a primate(chimpanzee), a feline (cat), or a canine (dog). Preferably, the subjectis human. The subject according to the invention may be in particular amale or a female.

According to the present invention, the subject suffers from obesity.

As used herein, the term “obesity”, “general obesity” or “overallobesity” refers to a medical condition in which excess body fat hasaccumulated to the extent that it may have an adverse effect on health,leading to reduced life expectancy and/or increased health problems.General obesity is typically determined by assessing the body mass index(BMI), a measurement which associates weight and height. In particular,people are defined as overweight if their BMI is between 25 kg/m² and 30kg/m², and obese when it is greater than 30 kg/m².

In the context of the invention, the subject has preferably a body massindex (BMI) higher than 30 kg/m², more preferably than 37.5 kg/m², oreven more preferably higher than 40 kg/m².

In the context of the invention, the term “abdominal obesity”, “centralobesity” or “belly fat” refers to obesity wherein there is a specificaccumulation of abdominal fat resulting in an increase in waist size.Typically, in abdominal obesity, visceral fat, also known as organ fator intra-abdominal fat, is located inside the peritoneal cavity, packedin between internal organs and torso, whereas, in general obesity,subcutaneous fat is found underneath the skin, and intramuscular fat isfound interspersed in skeletal muscle.

Abdominal obesity is typically determined just by looking at the nakedbody, or more specifically by taking waist and hip measurements. Theabsolute waist circumference (>102 centimeters (40 inches) in menand >88 centimeters (35 inches) in women) and the waist-hip ratio (>0.9for men and >0.85 for women) are both used as measures of abdominalobesity. Preferably, the expression “abdominal adiposity” according tothe invention refers to a waist circumference of more than 102 cm in menor of more than 88 cm in women.

Preferably, the subject according to the invention suffers fromnon-alcoholic fatty liver disease (NAFL disease) at the time ofsampling. More preferably, the subject according to the inventionsuffers from non-alcoholic steatohepatitis (NASH) at the time ofsampling.

“Non-alcoholic fatty liver disease” or “NAFL disease” is a term usedherein to describe the accumulation of fat in the liver of people whodrink little or no alcohol. In many cases, NAFL disease is linked toobesity. NAFL disease is common and, for most people, causes no signsand symptoms and no complications. But in some people with NAFL disease,the fat that accumulates can cause inflammation and scarring in theliver. This more serious form of NAFL disease is called non-alcoholicsteatohepatitis (NASH).

In the context of the invention, the expression “non-alcoholicsteatohepatitis” or “NASH” refers to a disease characterized by fataccumulation (steatosis) and inflammation. Steatosis results fromhepatic triglyceride accumulation. NASH can cause damage in the liverresulting in fibrosis.

In a particular embodiment, the subject has a body mass index (BMI)higher than 37.5 kg/m² and suffers from at least two metabolicco-morbidities selected from the group consisting of type 2 diabetes,hypertension and dyslipidemia.

As used herein, “diabetes” or “diabetes mellitus” denotes a metabolicdisorder in which the pancreas produces insufficient amounts of insulin,or in which the cells of the body fail to respond appropriately toinsulin thus preventing cells from absorbing glucose. As a result,glucose builds up in the blood. This high blood glucose level producesthe classical symptoms of polyuria (frequent urination), polydipsia(increased thirst) and polyphagia (increased hunger). The term“diabetes” includes type 1 diabetes, type 2 diabetes, gestationaldiabetes (during pregnancy) and other states that cause hyperglycemia.

Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus(NIDDM) and adult-onset diabetes, is associated with predominant insulinresistance and thus relative insulin deficiency and/or a predominantlyinsulin secretory defect (or insulinopenia) with insulin resistance.More specifically, type 2 diabetes may be associated either with (i) apredominant insulin resistance with a moderate insulinopenia or with(ii) a moderate insulin resistance with a predominant insulinopenia.

As used herein, the term “hypertension” also referred to as “high bloodpressure”, “HTN” or “HPN”, denotes a medical condition in which theblood pressure is chronically elevated. In the context of the invention,hypertension is preferably defined by systolic/diastolic blood pressureof at least 140/90 mmHg or being on antihypertensive medication.

As used herein, the term “dyslipidemia” denotes an elevation of plasmacholesterol, triglycerides or both, or a low high-density lipoproteinlevel that may contribute to the development of atherosclerosis.

In a particular embodiment, the subject is free of known systemicdisease such as rheumatoid arthritis, hypertension or systemic lupuserythematosus, serious chronic illness such as cardiovascular disease orcancer, and/or ethanol intake superior to 20 grammes per day.

In another particular embodiment, the subject according to the inventiondid not display infection symptom(s) during the month precedingsampling. Accordingly, the subject according to the invention preferablydisplays a plasma baseline C reactive protein concentration lower than10 mg/l and/or does not present an abundant leukocyturia and/or does nottake antiviral therapy.

As used herein, the term “C reactive protein” or “CRP” refers to aprotein which is a member of the class of acute-phase reactants, as itslevels rise dramatically during inflammatory processes occurring in thebody. As known from the skilled person, CRP is typically a 224-residueprotein with a monomer molar mass of 25 kDa, encoded by the CRP gene.

As used herein, the term “leukocyturia” refers to the presence ofleukocytes in the urine of the subject. In particular, an abundantleukocyturia corresponds typically to the presence of more than 10leukocytes/mm³ in the urine.

Bacterial 16S rDNA

In the context of the invention, the expressions “16S rDNA” and “16Sribosomal DNA” are used indifferently and refer to the gene encoding the16S ribosomal RNA constituted of about 1500 nucleotides, which is themain component of the small prokaryotic ribosomal subunit (30S). 16SrDNA is highly conserved among bacteria. The reference Escherichia coli16S rDNA gene sequence corresponds to SEQ ID NO: 1 (called rrs). In thecontext of the invention, 16S rDNA refers to any sequence correspondingto SEQ ID NO: 1 in other bacterial strains.

Biological Sample

As used herein, the term “biological sample” means a substance ofbiological origin. Examples of biological samples include blood andcomponents thereof such as serum, plasma, platelets, buffy coat(leucocytes), erythrocytes, urine, saliva, fecal water and tissues suchas adipose tissues, hepatic tissues, pancreatic tissues and the like.Preferably, a biological sample according to the present invention is ablood, serum, plasma, buffy coat, urine, adipose tissue or hepatictissue sample. More preferably, the biological sample is selected fromthe group consisting of blood, buffy coat, serum and plasma sample. Thebiological sample according to the invention may be obtained from thesubject by any appropriate means of sampling known from the skilledperson.

In Vitro Method for Diagnosing Liver Fibrosis

The present invention concerns an in vitro method for diagnosing liverfibrosis in a subject suffering from obesity, said method comprising thesteps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), diagnosing liverfibrosis in the subject suffering from obesity.

Liver biopsy is the standard for diagnosing liver fibrosis and fordiagnosing the underlying liver disorder causing fibrosis. However,liver biopsy is invasive. Therefore, liver biopsy should not be donesystematically, but rather to confirm the presence of liver fibrosis ina subject.

Thus the present invention also concerns an in vitro method forselecting a subject suffering from obesity for liver biopsy, said methodcomprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo liver biopsy.

Preferably, the concentration of bacterial 16S rDNA, or the ratio of thequantity of bacterial 16S rDNA to the quantity of total DNA (noted “16SrDNA/total DNA ratio”), is measured by polymerase chain reaction (PCR),more preferably by quantitative PCR (qPCR), most preferably by real-timeor real-time quantitative PCR (RT-PCR or RT-qPCR).

As used herein, “real-time PCR”, “real-time quantitative PCR”,“real-time polymerase chain reaction” or “kinetic polymerase chainreaction” refers to a laboratory technique based on the polymerase chainreaction, which is used to amplify and simultaneously quantify atargeted DNA molecule. It enables both detection and quantification (asabsolute number of copies or relative amount when normalized to DNAinput or additional normalizing genes) of a specific sequence in asample. Two common methods of quantification are the use of fluorescentdyes that intercalate with double-stranded DNA, and modified DNAoligonucleotide probes that emit fluorescence when hybridized with acomplementary DNA.

In the context of the invention, the real-time PCR is preferablyperformed using the universal forward and reverse primers eubac-F(5′-TCCTACGGGAGGCAGCAGT-3′ SEQ ID NO: 2) and eubac-R(5′-GGACTACCAGGGTATCTAATCCTGTT-3′ SEQ ID NO: 3). Typically, theamplification is performed using 2 μl of DNA in a total reaction volumeof 12.5 μl, using for instance Sybr Green RT-qPCR technologies,typically with the following cycle: hold stage of 10 min at 95° C., then40 cycles of 15 sec at 95° C., 1 min at 63° C. and 1 min at 72° C.Typically, specificity of the qPCR reaction is assessed by analysis of apost-PCR melting curve for example between 60° C. and 95° C.

Specifically, the inventors demonstrated that the concentration ofbacterial 16S rDNA (for instance the number of copies per ml of blood),or the 16S rDNA/total DNA ratio (for instance the number of copies perμg of total DNA), is significantly higher in biological samples ofsubjects with liver fibrosis.

The 16S rDNA concentration is preferably measured by real-time PCR,preferably using the universal forward and reverse primers eubac-F(5′-TCCTACGGGAGGCAGCAGT-3′ SEQ ID NO: 2) and eubac-R(5′-GGACTACCAGGGTATCTAATCCTGTT-3′ SEQ ID NO: 3). Typically, theamplification is performed using 2 μl of DNA in a total reaction volumeof 12.5 μl, using for instance Sybr Green RT-qPCR technologies,typically with the following cycle: hold stage of 10 min at 95° C., then40 cycles of 15 sec at 95° C., 1 min at 63° C. and 1 min at 72° C.Typically, specificity of the qPCR reaction is assessed by analysis of apost-PCR melting curve for example between 60° C. and 95° C.

In a particular embodiment, the method of diagnosing liver fibrosis inan obese subject as defined above further comprises a step a2) ofcomparing the measured bacterial 16S rDNA concentration, or the 16SrDNA/total DNA ratio, with a threshold value.

Preferably, when the bacterial 16S rDNA concentration is measured atstep a2), the threshold value corresponds to a normal concentration ofbacterial 16S rDNA. Also preferably, when the 16S rDNA/total DNA ratiois measured at step a2), the threshold value corresponds to a normalratio of 16S rDNA/total DNA.

As intended herein a “normal concentration” of bacterial 16S rDNA,respectively a “normal ratio” of 16S rDNA/total DNA, means that theconcentration of 16S rDNA, respectively the 16S rDNA/total DNA ratio, inthe biological sample is within the norm cut-off values for that gene.The norm is dependent on the biological sample type and on the methodused for measuring the concentration of 16S rDNA, respectively the 16SrDNA/total DNA ratio, in the biological sample. In particular, thethreshold value may be the concentration of bacterial 16S rDNA,respectively the 16S rDNA/total DNA ratio, that gives a negativepredictive value and a positive predictive value superior to 80%,preferably superior to 85%, more preferably superior to 90%, even morepreferably superior to 95% in the targeted population.

As used herein, the term “targeted population” refers to a populationconstituted of subjects who share certain biological parameters such ase.g. gender, age group, or certain environmental parameters such as e.g.geographical region.

Preferably, in the methods according to the invention, the thresholdvalue of the 16S rDNA copies/μl of buffy coat is between 460 and 760,still preferably between 550 and 720, most preferably of 675, saidthreshold value being preferably used when the concentration ofbacterial 16S rDNA is measured by real-time PCR, preferably using theuniversal forward and reverse primers eubac-F(5′-TCCTACGGGAGGCAGCAGT-3′, SEQ ID NO: 2) and eubac-R(5′-GGACTACCAGGGTATCTAATCCTGTT-3′, SEQ ID NO: 3). Typically, theamplification is performed using 2 μl of DNA in a total reaction volumeof 12.5 μl, using for instance Sybr Green RT-qPCR technologies,typically with the following cycle: hold stage of 10 min at 95° C., then40 cycles of 15 sec at 95° C., 1 min at 63° C. and 1 min at 72° C.Typically, specificity of the qPCR reaction is assessed by analysis of apost-PCR melting curve for example between 60° C. and 95° C.

Preferably, in the methods of the invention, it is further determinedwhether the measured concentration of bacterial 16S rDNA, or themeasured 16S rDNA/total DNA ratio, is increased or decreased compared tothe threshold value according to the invention. Still preferably, in themethods of the invention, it is further determined the level of increaseor decrease of the measured concentration of bacterial 16S rDNA, or ofthe measured 16S rDNA/total DNA ratio, compared to the threshold valueaccording to the invention.

As used herein, the expression “level of increase” means the percentageof increase of the measured concentration of bacterial 16S rDNA, or ofthe measured 16S rDNA/total DNA ratio, compared to the threshold valueaccording to the invention or the number of fold of increase of themeasured concentration of bacterial 16S rDNA, or of the measured 16SrDNA/total DNA ratio, compared to the threshold value according to theinvention.

Preferably, when the measured concentration or ratio is increasedcompared to the threshold value, its value is significantly higher thanthe threshold value.

Also preferably, when the measured concentration or ratio is decreasedcompared to the threshold value, its value is significantly lower thanthe threshold value.

The inventors specifically demonstrated that the increase of the 16SrDNA concentration (for instance the number of copies per ml of buffycoat) in the biological sample of a subject compared to the thresholdvalue enabled diagnosing a liver fibrosis with a very high significance,typically with a sensitivity of 91% and a specificity of 73% or apositive predictive value of 88% and negative predictive value of 86%.

Accordingly, in the methods for diagnosing liver fibrosis according tothe invention, a concentration or a ratio measured in step a1) which ishigher than the threshold value is preferably indicative of the presenceof liver fibrosis in the subject.

Also in the methods for diagnosing liver fibrosis according to theinvention, a concentration or a ratio measured in step a1) which islower than the threshold value is preferably indicative of an absence ofliver fibrosis in the subject.

Preferably, in the methods for selecting a subject suffering fromobesity for liver biopsy according to the invention, the subject isselected to undergo liver biopsy if the concentration or ratio measuredin step a1) is higher than the threshold value.

According to another aspect, the invention relates to an in vitro methodfor selecting a subject suffering from obesity for treatment regimentargeting liver fibrosis and/or its complications, said methodcomprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo treatment regimen targetingliver fibrosis and/or its complications.

A “treatment regimen targeting liver fibrosis and/or its complications”may for instance be increased surveillance for liver cancer, increasedsurveillance for oesophageal varices, or drug treatment.

As used herein, “drug treatment” or “drug treatment targeting liverfibrosis and/or its complications” may for instance refer to treatmentwith a pancreatic lipase inhibitor, a PPARgamma agonist, a leptinanalogue, a probiotic or a prebiotic.

In a particular embodiment, the method of diagnosing liver fibrosis inan obese subject as defined above, or the method for selecting a subjectsuffering from obesity for treatment regimen targeting liver fibrosisand/or its complications as defined above, further comprises a step c)of submitting the subject to a treatment regimen targeting liverfibrosis and/or its complications, if liver fibrosis has been diagnosedin step b).

Methods of Monitoring

The inventors demonstrated that the concentration of bacterial 16S rDNA,or the bacterial 16S rDNA/total DNA ratio, in the biological sample of asubject suffering from fibrosis may be useful in the diagnosis of liverfibrosis. Subjects who have been diagnosed as suffering from liverfibrosis may further benefit from an appropriate monitoring of liverfibrosis or their complications.

Accordingly, another aspect of the present invention is a method formonitoring an obese subject suffering from liver fibrosis and/orcomplications thereof, said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject as defined in the above section “Invitro method for diagnosing liver fibrosis”;

b) based on the result of the measurement in step a1), diagnosing liverfibrosis in the subject suffering from obesity as defined in the abovesection “In vitro method for diagnosing liver fibrosis”; and

c) monitoring the subject for complications of liver fibrosis, if liverfibrosis has been diagnosed in step b).

In the context of the invention, the expression “monitoring a subjectfor complications of liver fibrosis” means submitting said subject toclinical care including for instance ultrasounds for detection of livercancer, or gastric fibroscopy for detection of varices of the esophagus.Such clinical care is well-known for the skilled person.

Methods of Treatment

According to another aspect, the invention relates to a method fortreating a subject suffering from obesity and from liver fibrosis and/orits complications, said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject;

b) based on the result of the measurement in step a1), diagnosing liverfibrosis in the subject suffering from obesity; and

c) submitting the subject selected in step b) to a treatment regimentargeting liver fibrosis and/or its complications.

The invention further pertains to a method for treating a subjectsuffering from obesity and from liver fibrosis and/or its complications,said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject;

b) based on the result of the measurement in step a1), selecting thesubject suffering from obesity to undergo drug treatment targeting liverfibrosis and/or its complications; and

c) administering to the subject selected in step b) a drug treatmenttargeting liver fibrosis and/or its complications.

In particular, the drug treatment targeting liver fibrosis and/or itscomplications is as defined in the section “in vitro method fordiagnosing liver fibrosis”.

The invention also pertains to in vitro method for monitoring theresponsiveness of a patient suffering from obesity and from liverfibrosis and/or its complications to a drug treatment targeting liverfibrosis and/or its complications, said method comprising the steps of:

a1) measuring the concentration of bacterial 16S rDNA, or the ratio ofthe quantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of the subject; and

b) based on the result of the measurement in step a1), monitoring theresponsiveness of the patient to said drug treatment.

The expression “monitoring the responsiveness of a patient to a drugtreatment targeting liver fibrosis and/or its complications” may forinstance mean adapting the drug treatment. Preferably, “monitoring theresponsiveness of a patient to a drug treatment targeting liver fibrosisand/or its complications” means changing the drug used to treat thepatient, or increasing or reducing the dose, the administrationfrequency, or changing the administration route of the drug treatment.

Methods of Screening

The present invention also pertains to method for screening a probiotic,a prebiotic, a chemical compound or a biological compound suitable forpreventing and/or treating liver fibrosis, comprising the steps of:

measuring the concentration of bacterial 16S rDNA, or the ratio of thequantity of bacterial 16S rDNA to the quantity of total DNA, in abiological sample of an obese subject suffering from liver fibrosis whohas been treated with the candidate probiotic, prebiotic, chemicalcompound or biological compound; and

comparing said concentration, or said ratio, with that of a controlobese subject suffering from liver fibrosis who has not been treatedwith said candidate probiotic, prebiotic, chemical compound orbiological compound;

wherein a higher concentration, or a higher ratio, measured in thebiological sample of the control subject than in the biological sampleof the subject treated with the candidate probiotic, prebiotic, chemicalcompound or biological compound, indicates that said candidateprobiotic, prebiotic, chemical compound or biological compound issuitable for preventing and/or treating liver fibrosis.

As used herein, the term “probiotics” denotes dietary supplements andlive microorganisms containing potentially beneficial bacteria oryeasts. According to the currently adopted definition by FAO/WHO,probiotics correspond to live microorganisms which when administered inadequate amounts confer a health benefit on the host. Examples ofprobiotics according to the invention include bacterial strains of thegenera bifidobacterium, lactobacillus, bacteroides or of the classfusobacteria.

As used herein, the term “prebiotics” denotes a non-digestible foodingredient that beneficially affects the host by selectively stimulatingas a substrate the growth and/or activity of one or a limited number ofbacteria in the intestine, in particular in the colon, and thus improveshost health.

In the context of the invention, “prebiotics” encompass isolated orpurified prebiotics as well as natural prebiotics present in dietarysupplements.

In the context of the invention, “probiotics”, encompass isolated orpurified probiotics as well as natural probiotics present in dietarysupplements.

As used herein, the term “chemical or biological compound” encompasseschemically synthetized compounds and compounds of biological originwhich have an effect on the growth, metabolism, the survival of bacteriaand/or their passage through the intestinal barrier. In particular,chemical or biological compounds according to the invention includemolecules which modify the bacterial flora of the digestive tract and/orwhich modify the migration of bacteria through the digestive tractand/or which modify the permeability of the intestinal epithelialbarrier. Examples of chemical or biological compounds of the inventioninclude bactericides, antibiotics, as well as compounds acting onepithelial intercellular tight junctions, microvillies, cell coat,and/or intestinal epithelial cells.

The control subject which has not been treated may be a subjectunrelated to the subject receiving the candidate prebiotic, probiotic orchemical or biological compound, or the same subject before treatmentwith the candidate prebiotic, probiotic or chemical or biologicalcompound.

The invention will be further illustrated by the following examples.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the sequence of the reference Escherichia coli 16SrDNA gene.

SEQ ID NO: 2 shows the sequence of the universal rDNA forward primereubac-F.

SEQ ID NO: 3 shows the sequence of the universal rDNA reverse primereubac-R.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the distribution of the level of 16S bacterial DNA in theblood of 37 patients as measured by a 16S rDNA qPCR assay.

FIG. 2 shows the concentration of blood bacterial 16S rDNA in patientswith or without fibrosis, as measured by a 16S rDNA qPCR assay.

FIG. 3 shows the concentration of blood bacterial 16S rDNA in patientswith no fibrosis or with fibrosis at various stages, as measured by a16S rDNA qPCR assay.

FIG. 4 shows the ROC curve for the diagnosis of liver fibrosis.

EXAMPLE 1

Materials and Methods

Population

A cross sectional study was carried out in the Florinash cohort(http://www.florinash.org/). Included patients had severe obesity(BMI>40 or BMI>37.5 plus two metabolic co-morbidities such as Type 2diabetes, hypertension and dyslipidemia, according to ATPIII) and hadbeen oriented to gastric bypass surgery, once that appropriate lifestyleinterventions had failed, to reduce body weight and reducecardiovascular risk. Exclusion criteria were systemic disease, infectionin the previous month, serious chronic illness, >20 g ethanol intake perday. All patients for whom buffy coat fraction samples were availablewere analysed.

Determination of Blood 16S rDNA Gene Concentration

Total DNA from 100 μl buffy coat fraction were extracted using aSilica-membrane technology kit. Total DNA concentration was determinedusing the UV spectrometer Nanodrop 2000 (Thermo Scientific). Thebacterial DNA (16S rDNA) content was quantified by real-timequantitative PCR (ViiA™ 7 real time PCR System, Life technology) usingoptical grade 384-well plates. The qPCR reaction was performed using theuniversal rDNA forward and reverse primers eubac-F(5′-TCCTACGGGAGGCAGCAGT-3′, SEQ ID NO: 2) and eubac-R(5′-GGACTACCAGGGTATCTAATCCTGTT-3′, SEQ ID NO: 3). The amplification stepwas followed by a melting curve step to determine the specificity of theamplification product obtained. The amount of amplified DNA (16S rDNA)was expressed as raw data 2^(−ct) or normalized with a plasmid basedstandard scale of E. Coli 16S rDNA. The coefficient of variation(repeatability) of the method (0.24%) was determined using 30 replicatemeasurements.

Statistical Analysis

The characteristics of the participants who did and did not havefibrosis are shown and non-parametric Mann-Whitney's tests and Fisher'sexact tests were conducted on quantitative and categorical variablerespectively. Furthermore, the ROC curve for the diagnosis of liverfibrosis is presented.

Results

A bacterial 16S rDNA qPCR assay was performed with blood samples of the37 patients. This assay revealed, as shown on the distribution of FIG.1, that two different populations coexist in the cohort: one with a lowlevel and one with a high level of 16S bacterial DNA in the blood whichcorrelate with liver fibrosis status. Analysis of the correlationbetween fibrosis status of the patients and blood bacterial 16S rDNAconcentration showed a very significant increase of blood bacterial 16SrDNA with fibrosis (FIG. 2) which is remarkably correlated to the stageof the disease (FIG. 3). This result demonstrates that liver fibrosis isassociated in the cohort to a clear change in the amount of bacterial16S DNA in the blood which correlates with the severity of the disease.

As shown in FIG. 4, the area under the ROC curve for the diagnosis ofliver fibrosis is 0.87.

The characteristics of the population are presented in Table 1. 16S rDNAconcentration (copies/μl) was significantly higher in patients withliver fibrosis (Table 1). The distribution of 16S rDNA concentration wasshifted towards higher values in patients with liver fibrosis. At athreshold of 675 copies of 16S rDNA/μl of buffy coat, the negativepredictive value is 86%, the positive predictive value is 88%, thesensitivity of the test is 55% and the specificity is 96%. At athreshold of 281 copies of 16S rDNA/μl of buffy coat, the negativepredictive value is 95%, the positive predictive value is 56%, thesensitivity of the test is 91% and the specificity is 73%.

In conclusion, the results demonstrate that the determination of blood16S rDNA gene concentration enables diagnosing or ruling out liverfibrosis in obese patients.

TABLE 1 Characteristics of the study population Controls Cases (Nofibrosis) (Fibrosis) Total (n) Cases vs 26 11 controls Categoricalvariables n % n % p (Fisher) Men 5 2 1.00 Women 21 9 1.00 Smokingstatus* Never smoked 12 48.0 6 54.5 1.00 Former smoker 7 28.0 3 27.3Current smoker 6 24.0 2 18.2 Treated hypertension 11 42.3 6 54.5 0.72Treated diabetes 6 23.1 5 45.5 0.24 Treated dyslipidemia 6 23.1 2 18.21.00 p (Mann- Quantitative variables Mean SD Mean SD Whitney) Age(years) 46.2 8.9 48.1 9.3 0.56 Body mass index 44.7 6.7 41.9 6.5 0.17(kg/m²) Total cholesterol 193.5 34.2 188.5 28.1 0.66 (mg/dl) HDLcholesterol 47.5 10.5 44.2 5.5 0.27 (mg/dl) GPT (U/l) 25.2 19.29 24.366.454 0.17 GGT (U/l) 20.5 9.4 23.9 7.2 0.1 Glucose (mg/dl) 99.6 29.2112.5 37.4 0.39 C reactive protein 0.73 0.44 0.78 0.75 0.57 (mg/dl)*Hematocrit (%) 40.5 4.6 38.6 4.0 0.33 Leukocyte (10³/μl) 7.3 2.0 7.8 2.00.55 Neutrophil (10³/μl) 4.6 1.9 4.9 1.9 0.64 Blood 16S DNA 239.3 186.4652.6 285.4 0.0002 (copies/μl) GPT: Glutamic-Pyruvic Transaminase, GGT:Gamma-glutamyl transpeptidase, HDL: High-density lipoprotein. *Data ismissing for one patient.

The invention claimed is:
 1. A method for diagnosing and treating asubject suffering from obesity for a treatment regimen targeting liverfibrosis and/or complications from liver fibrosis, said methodcomprising a1) measuring the concentration of bacterial 16S rDNA, or theratio of the quantity of bacterial 16S rDNA to the quantity of total DNAin a biological sample of a subject; b) diagnosing liver fibrosis in thesubject suffering from obesity based on the result of the measurement ina1); and c) administering a drug treatment targeting liver fibrosisand/or complications from liver fibrosis to the subject.
 2. The methodaccording to claim 1, further comprising a2) comparing the concentrationor the ratio measured in a1) with a threshold value.
 3. The methodaccording to claim 2, wherein a concentration or a ratio measured in a1)which is higher than the threshold value is indicative of liver fibrosisin the subject.
 4. The method according to claim 1, wherein thebiological sample is selected from the group consisting of blood, serumand plasma sample.
 5. The method according to claim 1, wherein theconcentration or the quantities measured in a1) is/are measured byreal-time PCR.
 6. The method according to claim 1, wherein the subjectsuffers from non-alcoholic fatty liver disease (NAFL) at the time ofsampling.
 7. The method according to claim 1, wherein the subjectsuffers from non-alcoholic steatohepatitis (NASH) at the time ofsampling.
 8. The method according to claim 1, wherein the subject has abody mass index (BMI) higher than 37.5 kg/m² and suffers from at leasttwo metabolic co-morbidities selected from the group consisting of type2 diabetes, hypertension and dyslipidemia.
 9. The method according toclaim 1, wherein the subject has a body mass index (BMI) higher than 40kg/m².
 10. The method according to claim 1, wherein the subject did notdisplay infection symptom(s) during the month preceding sampling.
 11. Amethod for selecting a subject suffering from obesity for a treatmentregimen targeting liver fibrosis and/or complications from liverfibrosis, said method comprising a1) measuring the concentration ofbacterial 16S rDNA, or the ratio of the quantity of bacterial 16S rDNAto the quantity of total DNA in a biological sample from a subjectsuffering from obesity; b) diagnosing liver fibrosis in the subjectbased on the result of the measurement in a1); c) selecting the subjectto undergo a treatment regimen targeting liver fibrosis and/orcomplications from liver fibrosis; and d) administering a drug treatmenttargeting liver fibrosis and/or complications from liver fibrosis to thesubject.